Food Animal Health Research Program, Ohio Agricultural Research and Development Center, The Ohio State University, 1680 Madison Avenue, Wooster, OH, 44691, USA.
Arch Virol. 2014 Feb;159(2):257-66. doi: 10.1007/s00705-013-1806-4. Epub 2013 Aug 23.
Human noroviruses (HuNoVs) are a leading cause of acute gastroenteritis. Establishment of a cell culture system for in vitro HuNoV growth remains challenging. Replication of HuNoVs in human intestinal cell lines (INT-407 and Caco-2) that differentiate to produce microvilli in rotation wall vessel (RWV) three-dimensional cultures has been reported (Straub et al. in Emerg Infect Dis 13:396-403, 2007; J Water Health 9:225-240, 2011, and Water Sci Technol 67:863-868, 2013). We used a similar RWV system, intestinal cell lines, and the same (Genogroup [G] I.1) plus additional (GII.4 and GII.12) HuNoV strains to test the system's reproducibility and to expand the earlier findings. Apical microvilli were observed on the surface of both cell lines by light and electron microscopy. However, none of the cell types tested resulted in productive viral replication of any of the HuNoV strains, as confirmed by plateau or declining viral RNA titers in the supernatants and cell lysates of HuNoV-infected cells, determined by real-time reverse transcription PCR. These trends were the same when culture supplements were added that have been reported to be effective for replication of other fastidious enteric viruses in vitro. Additionally, by confocal microscopy and orthoslice analysis, viral capsid proteins were mainly observed above the actin filament signals, which suggested that the majority of viral antigens were on the cell surface. We conclude that even intestinal cells displaying microvilli were not sufficient to support HuNoV replication under the conditions tested.
人类诺如病毒（HuNoVs）是急性肠胃炎的主要病因。建立用于 HuNoV 体外生长的细胞培养系统仍然具有挑战性。已经报道了 HuNoVs 在人肠细胞系（INT-407 和 Caco-2）中的复制，这些细胞系在旋转壁容器（RWV）三维培养中分化产生微绒毛（Straub 等人，《新发传染病》13:396-403，2007；J Water Health 9:225-240，2011，和 Water Sci Technol 67:863-868，2013）。我们使用了类似的 RWV 系统、肠细胞系以及相同的（基因群[G]I.1）加额外的（GII.4 和 GII.12）HuNoV 株来测试该系统的重现性并扩展早期的发现。通过光镜和电子显微镜观察到两种细胞系表面都有顶端微绒毛。然而，在测试的任何细胞类型中，都没有导致任何 HuNoV 株的有效病毒复制，这通过实时逆转录 PCR 确定的 HuNoV 感染细胞上清液和细胞裂解物中病毒 RNA 滴度的平台或下降得到证实。当添加据报道对其他苛刻肠道病毒在体外复制有效的培养补充剂时，这些趋势也是相同的。此外，通过共聚焦显微镜和正交切片分析，病毒衣壳蛋白主要观察到在肌动蛋白丝信号之上，这表明大多数病毒抗原位于细胞表面。我们得出结论，即使是显示微绒毛的肠细胞在测试条件下也不足以支持 HuNoV 复制。
